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Objectives: While evaluating the validity of using normal human mucosal cells from the upper aerodigestive tract as diploid standards for DNA content studies of squamous cell cancer of head and neck by flow cytometry, pseudoaneuploidy was frequently detected. The purpose of this study was to further evaluate these DNA content abnormalities encountered in normal human mucosal cells and correlate them to physiological apoptosis., Study Design: Thirty-two specimens of upper areodigestive tract mucosa from 18 surgical resections, 11 fresh autopsies, and 3 buccal scrapings were examined for DNA content by flow cytometry., Results: Pseudoaneuploidy, which ranged from sub-G0/G1 peaks to hyperdiploid peaks with increased 90 degrees light scattering properties was found in 60% of these specimens. Fluorescent microscopic examination of the sorted DNA pseudoaneuploid cells demonstrated cells undergoing apoptosis., Conclusion: This unexpected pseudoaneuploidy in normal mucosal cells was a result of physiological apoptosis, a normal component of squamous differentiation., Ebm Rating: B-2.

Purpose: The objective of our investigation was to prospectively study what the implications of an unresponsive CD3 receptor are on clinical outcome in advanced-stage head and neck cancer patients., Experimental Design: Lymph node mononuclear cells were purified from cancer patients and stimulated with immobilized anti-CD3 in vitro for 8 days. Two populations were identified, nonresponders (NRs) with [(3)H]thymidine-counts per min (cpm) <3500 and responders (Rs) with cpm >or=3500. NRs and Rs were prospectively followed for a minimum of 24 months, and clinical outcomes were compared. Postoperative complications, length of hospitalization, toxicities associated with chemotherapy or radiation therapy, survival, and disease-free status were measured., Results: Twenty-six patients were followed, of which 19 Rs [[(3)H](X) = 37,819 +/- 24,979 cpm (mean proliferative count +/- SD)] and 7 NRs ([(3)H](X) = 1,375 +/- 1,102 cpm) were identified. There were no phenotypic differences in lymph node T-cell subpopulations (CD3, CD4, CD8, CD28, CD45RO) between groups. There was a 71% (5/7) incidence of recurrent cancer in NRs compared with 16% (3/19) in Rs; the median disease-free interval was significantly less in NRs (P = 0.03). The risk ratio of Rs to develop a recurrent cancer was 0.237 (95% confidence interval, 0.057-0.994), much less than for NRs., Conclusions: Patients with an unresponsive CD3 receptor as measured by in vitro response to anti-CD3 monoclonal antibodies had a significantly higher incidence of recurrent cancer. Analyses using Cox proportion hazards models demonstrated that CD3 response was the single greatest predictor of reduced disease-free interval. This is the first prospective study to confirm the importance of regional lymph node mononuclear cell CD3 receptor function in head and neck squamous cell carcinoma patients for tumor control.

Objectives: To test whether T-cell CD3 responses are altered in patients with advanced-stage head and neck squamous cell carcinoma (HNSCC) and whether anti-CD3/anti-CD28 (alphaCD3/alphaCD28) bead stimulation could reverse CD3 unresponsiveness., Design: Anti-CD3 (alphaCD3) monoclonal antibody immobilized on tissue culture plastic was used to stimulate lymph node mononuclear cells (LNMCs) and peripheral blood mononuclear cells (PBMCs) from patients with advanced-stage HNSCC. Proliferation, T-cell phenotype, and cytokines were measured during 8-day in vitro stimulation. Immune-enhancing properties of alphaCD3/ alphaCD28 beads were also tested on LNMCs and PBMCs. Cytotoxicity of bead-activated T cells (ATCs) was measured against autologous and allogeneic HNSCC., Results: Six patients were nonresponders to alphaCD3 stimulation defined by tritium (3H) incorporation of less than 3500 cpm, whereas 11 patients were responders with 3H incorporation of 3500 cpm or more. Responders produced higher levels of interleukin (IL)-12 and interferon gamma (IFN-gamma) after alphaCD3 stimulation than nonresponders. No phenotypic or clinical differences were identified between groups. Stimulation with alphaCD3/alphaCD28 beads enhanced IFN-gamma and IL-2 produced by both groups. Bead ATCs were generated from PBMCs of patient 11 in the responder group and lysed (+/- SD) 100% +/-1% of autologous tumor and 49% +/-1% of allogeneic tumor. Bead ATCs from LNMCs of this patient lysed 58%+/-1% of autologous tumor and 63%+/-1% of allogeneic tumor., Conclusions: A subpopulation of patients with HNSCC who are nonresponders to alphaCD3 stimulation has been identified, showing reduced proliferation and IL-12 and IFN-gamma secretion. Nonresponders stimulated with alphaCD3/alphaCD28 beads reversed immune unresponsiveness and induced a type 1 cytokine response. Bead-generated ATCs from patient 11 in the responder group lysed autologous and allogeneic HNSCC in vitro, suggesting a possible effective immunotherapeutic modality in the treatment of HNSCC.

Purpose: Define radiological and histological features in which patients with head and neck cancer would benefit from a carotid artery resection. Resection of the carotid artery has been advocated for local control of advanced squamous cell carcinoma of the head and neck. To provide appropriate preoperative counseling and optimize the utilization of resources, the criteria for patient selection has to be defined., Methods: Thirty-four patients underwent carotid artery resection based on the clinical impression of tumor fixation. Eighteen and 28 patients were evaluated using computed tomography (CT) and histological analysis, respectively. The distance between the tumor cells and external elastic lamina was measured. CT scans were examined to determine the circumference of tumor attachment around the carotid artery., Results: Clinical assessment predicted tumor within 1.8 mm of the carotid artery in 68% of cases. The overall survival for patients with tumor greater than 1.8 mm (N = 9) was better than that of patients with less (N = 19) than 1.8 mm (33.3% vs. 5.3%; median 24 versus 9 mo, P = .0899). Three of six patients (50%) with less than 180 degrees circumference tumor attachment had tumor within 1.8 mm from the external elastic lamina. Eight of twelve patients (67%) with tumors encompassing more than 180 degrees of the artery wall had tumor within 1.8 mm from the external elastic lamina. The overall survival rates for patients with tumor attachment greater and less than 180 degrees were 8.3% and 33%, respectively., Discussion: Tumor invasion into the carotid artery was the strongest predictor of outcome. Clinical assessment was as predictive as CT for tumor invasion. If tumor involvement of the carotid artery is less than 180 degrees, peeling the tumor is an alternative to carotid artery resection.

Objective: To test whether anti-CD3/anti-CD28 (alphaCD3/alphaCD28) monoclonal antibodies could be coated on surgical suture and used to enhance T-cell immune function in patients with advanced-stage head and neck squamous cell carcinoma (HNSCC)., Design: AlphaCD3/alphaCD28 monoclonal antibodies at varying concentrations and ratios were coated on surgical sutures and tested on peripheral blood mononuclear cells from normal donors to identify the optimal stimulating condition. Immune-enhancing properties of alphaCD3/alphaCD28 monoclonal antibody suture were tested on peripheral blood mononuclear cells and regional lymph node mononuclear cells isolated from patients with advanced HNSCC and on normal donor peripheral blood mononuclear cells. Proliferation, T-cell phenotype, and cytokines were measured during 8-day in vitro stimulation with alphaCD3/alphaCD28 suture and compared with alphaCD3/alphaCD28-coated tissue culture plastic, a previously recognized carrier., Results: Optimal stimulation was observed with monofilament nylon incubated with alphaCD3/alphaCD28, 2 microg/mL, at a 1:1 ratio for 18 hours at 37 degrees C. Strong proliferation of peripheral blood mononuclear cells and lymph node mononuclear cells in patients with HNSCC was induced by alphaCD3/alphaCD28 suture. There was no difference in maximal proliferation between alphaCD3/alphaCD28 plastic and suture. On day 6 after alphaCD3/alphaCD28 suture stimulation, T-cell subpopulations expressing CD3, CD4, CD8, CD28, and CD45RO were enhanced. Suture stimulation significantly enhanced interleukin 2 secretion when compared with plastic stimulation (P = .01). Both alphaCD3/alphaCD28 suture and plastic stimulated interferon gamma secretion., Conclusions: To our knowledge, this study is the first to report the modification of surgical suture to create an immunomodulant. AlphaCD3/alphaCD28-coated suture expanded T cells from patients with HNSCC and induced a T(H)1 immune response, which may be a useful therapeutic tool in the treatment of HNSCC and other diseases.

The progression of normal squamous epithelium to a malignant metastatic phenotype may depend on cellular genetic events and the failure of host mechanisms. Intermediate biomarkers are needed to more effectively identify and quantify malignant progression and develop the potential for specific treatments and prevention strategies. The nuclear matrix is the RNA-protein scaffold of the nucleus, which controls in part nuclear shape, DNA organization, and DNA function. Nuclear matrix proteins in all previously studied cell types show a common set of nuclear matrix proteins and a subset of tissue- and cell type-specific proteins. In every system studied to date, the nuclear matrix has been demonstrated to undergo quantifiable alterations in its protein composition with transformation to the malignant phenotype. The loss and gain of nuclear matrix proteins are being investigated as biomarkers for malignant transformation in breast, colon, and prostate carcinoma. We have investigated nuclear matrix protein composition in laryngeal and oral cavity primary squamous cell tumors and metastatic cervical lymph nodes. Laryngeal carcinoma demonstrated the gain of two specific nuclear matrix proteins in comparison with noncancerous squamous epithelium. Squamous cell carcinoma matrixes demonstrate greater heterogeneity than do previously studied adenocarcinoma matrixes, and yet they display specific matrix proteins that may represent important potential biomarkers.

Experimental research involving human solid tumors often requires single cell suspensions of high yield that are representative of the tissue of origin and in which the cellular property of interest is preserved. This is particularly necessary for the determination of DNA ploidy by flow cytometry. Mechanical dissaggregation and proteolytic enzyme digestion are the most commonly employed dissociation techniques for solid tumors. Comparative testing of techniques is often not performed. Mechanical and proteolytic enzyme dissociation techniques were comparatively tested in 77 human squamous cell cancers of the head and neck (SCCHN) and 25 human colon cancers for cellular yield, dye exclusion viability, quality, and morphology of DNA histograms, and the presence and proportion of DNA aneuploid subpopulations. Significant and consistent DNA aneuploid subpopulation losses were noted in mechanical preparations of SCCHN and enzymatic preparations of colon cancers. The frequency of SCCHN specimens with DNA aneuploid subpopulations was underestimated by 52% in mechanical cell suspensions, and the proportion of DNA aneuploid cells was diminished in an additional 30% of the specimens. Conversely, the frequency of specimens with DNA aneuploid subpopulations was underestimated by 38% in cell suspensions from enzymatically dissociated human colon cancer and their proportion diminished in an additional 50% of the specimens. Incubations of human colon cancers with three commonly employed proteolytic enzymes demonstrated a progressive loss of DNA aneuploid subpopulations as a function of enzyme concentration and incubation time. This is a serious potential source of error in the flow cytometric determination of DNA ploidy in human solid tumors, and may contribute to the diversity of results obtained and occasional contradictory conclusions reached in such studies.

Treatment of patients with squamous cell carcinoma (SCC) of an unknown primary localized to the neck is still controversial, particularly regarding advanced disease. We reviewed 41 such patients treated with surgery and/or radiotherapy (RT) (n = 25) or with combined modality treatment including chemotherapy (CH) (n = 16). The male to female ratio was 28 to 13, and the median age was 58 years (range, 32 to 94 years). There were 27 (66%) patients with poorly differentiated SCC and 8 with moderately differentiated or well-differentiated cancer. Twenty-three (56%) patients had N3 disease, 16 (39%) had N2, and 2 had N1. The majority of N3 patients have been treated with CH and RT (n = 12) or with RT alone (n = 9). The combined CH-RT was well tolerated, with no life-threatening toxicity. The complete response (CR) to CH-RT was 81% (11 patients have no evidence of disease [NED] currently). The median survival time of this group was 37+ months. Of the 25 patients who had surgery and/or RT as their first planned treatment, 7 (28%) have NED currently. The median survival time of this group was 24 months. Patients with N3 disease who received CH had a higher CR rate and a longer survival time as compared with those treated with surgery and/or RT, despite a higher (N3) stage of disease. These findings warrant further investigation in randomized cooperative studies.

DNA analysis is currently being applied to many solid tumors to determine DNA content and synthesis phase fraction. We compared DNA content measured by flow cytometry with multiple histologic parameters in 155 squamous cell carcinomas of the upper aerodigestive tract. Abnormal DNA content (aneuploidy) was identified in 107 (69%) of the neoplasms. Abnormal DNA content or aneuploidy correlated with above-median synthesis phase fraction and increased frequency of mitotic figures. Other histologic features predicting aggressive tumor behavior also correlated with abnormal DNA content and included small cords or single cell pattern of tumor invasion, high nuclear grade, and decreased stromal or desmoplastic response to the invading squamous cell carcinoma.

The immunohistologic distribution of the basement membrane components, type IV collagen and laminin, was evaluated in 57 alcohol-fixed, paraffin-embedded tissue sections which demonstrated immunostaining patterns with antigen preservation similar to that of frozen tissue sections. Normal and hyperplastic squamous mucosa, a spectrum of intraepithelial neoplasia, and invasive squamous cell carcinomas of the upper aerodigestive tract were evaluated by this technique. Prominent and continuous basement membrane staining characterized normal and reactive hyperplastic squamous mucosa. The basement membrane varied greatly with epithelial dysplasia and was usually prominent and continuous in mild to moderate dysplasias. In severe dysplasia/carcinoma in situ, the basement membrane was often thinned and occasionally discontinuous. The distribution of basement membrane in invasive carcinomas was also varied. Basement membrane was usually present in invasive tumors with well-defined tumor host borders and cohesive patterns of stromal invasion which were interpreted as foci of histologic differentiation. In contrast, invasive carcinomas with irregular cords or single tumor cells distributed through the host stroma invariably lacked basement membrane at the tumor-stromal interface; this was interpreted as a decreased expression of histologic differentiation. We conclude that 1) severe intraepithelial neoplasia is often associated with irregularities of basement membrane and that the absence of basement membrane does not necessarily define invasive cancer; 2) immunolocalization of basement membrane in invasive carcinomas is common in areas displaying histologic differentiation; and 3) the association of basement membrane distribution and histologic pattern of tumor invasion suggests that squamous cell carcinomas are capable of undergoing focal histologic differentiation after invasion has occurred.